Vector-averaged gravity regulates gene expression of receptor activator of NF-κB (RANK) ligand and osteoprotegerin in bone marrow stromal cells via cyclic AMP/protein kinase A pathway

Bone loss due to unloading of the skeleton may be caused by an acceleration of osteoclastic bone resorption as well as a decline of osteoblastic bone formation. Recently, two molecular species that play important roles in osteoclastogenesis were discovered: (i) the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ligand/osteoclast differentiation factor induces osteoclastogenesis; and (ii) the OPG/osteoclastogenesis inhibitory factor potently inhibits osteoclastogenesis. To investigate the effects of gravity on gene expression of RANKL and OPG, a mouse bone marrow-derived stromal cell line, ST2, was cultured on a single axis clinostat, which generates a vector-averaged gravity environment. Northern blot analysis revealed that RANKL mRNA was increased, whereas that of OPG decreased. The clinostat culture also caused an increase in intracellular cyclic (cAMP) level. Both forskolin and dibutyryl-cAMP mimicked the regulation of RANKL and OPG transcription in clinostat culture. These modulations of gene expression in clinostat culture were blocked by a protein kinase A (PKA) inhibitor, H89, but not by a cyclooxygenase inhibitor, indomethacin. The enhancement of RANKL gene expression under clinostat culture and its inhibition by H89 were confirmed by a reporter assay with the murine RANKL 5′-flanking region. These results suggest that modulations of RANKL and OPG expression in stromal cells might be one of the causes of bone loss during skeletal unloading. An elevation of intracellular cAMP level caused through an as yet undetermined pathway is involved in modulation of RANKL and OPG expression during clinostat culture.